MHC class II antigen presentation by intestinal epithelial cells fine-tunes bacteria-reactive CD4 T cell responses
MHC class II antigen presentation by intestinal epithelial cells fine-tunes bacteria-reactive CD4 T cell responses
Lamina propria lymphocytes were isolated from C57BL/6 mice at 2 weeks of Helicobacter hepaticus + anti-IL10R colitis and 2.3x106 cells/ml were seeded in complete RPMI for 20h. Supernatants from cells of 7 mice were pooled and frozen at −80°C. Colonic organoids were seeded for stimulation at p4, p6 and p7. 24h prior to stimulation full medium was reduced to organoid medium containing 1% Wnt3a. Colonic organoids were stimulated with 10% control conditioned media or inflammatory conditioned media for 24h prior to lysis and RNA isolation (Qiagen RNeasy). Quadruplicates were sequenced. Organoids were cultured as previously described.1 Colonic crypts were isolated from longitudinal opened and cleaned colon tissue of Atg16l1fl/fl mice by 2mM EDTA in PBS incubation for 90min at 4°C. The tissue was vigorously resuspended in PBS/10% FCS and the supernatant containing the crypts filtered through a 100μm cell strainer and pelleted. Crypts were seeded in 20μl Matrigel/well (Corning) in a 24-well plate. After polymerisation of the Matrigel, 400ul of complete organoid medium was added [Advanced Dulbecco's Modified Eagle Medium (DMEM)/F12 supplemented with 1% Pen/Strep, 10mM HEPES, 1X GlutaMax, 50ng/ml mEGF, 1× N2, 1× B27 (all from Invitrogen), 1mM N-acetylcysteine (NAC), 10μM RhoK-inhibitor (Y-27632 dihydrochloride monohydrate) (both from Sigma) and conditioned medium containing Wnt3a (20%), Noggin (20%) or R-spondin (10%)]. The conditioned media were generated from Rspol-Fc Hek 293T cells (kind gift from Dr. Calvin Kuo, Stanford) and Nog-Fc Hek 293T cells (kind gift from Dr. Gjis van den Brink, Amsterdam) and Wnt3A L cells (ATCC CRL-2647). The medium was changed every 2-3d and organoids were expanded over several passages before they were used for experiments with indicated stimuli. Ref: 1 Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009). Qiagen Rneasy Kit Illumina Truseq mRNA kit